Radiotolerance of N-cycle bacteria and their transcriptomic response to low-dose space-analogue ionizing irradiation.

The advancement of regenerative life support systems (RLSS) is crucial to allow long-distance space travel. Within the Micro-Ecological Life Support System Alternative (MELiSSA), efficient nitrogen recovery from urine and other waste streams is vital to produce liquid fertilizer to feed food and oxygen production in subsequent photoautotrophic processes. This study explores the effects of ionizing radiation on nitrogen cycle bacteria that transform urea to nitrate. In particular, we assess the radiotolerance of Comamonas testosteroni, Nitrosomonas europaea, and Nitrobacter winogradskyi after exposure to acute γ-irradiation. Moreover, a comprehensive whole transcriptome analysis elucidates the effects of spaceflight-analogue low-dose ionizing radiation on the individual axenic strains and on their synthetic community o. This research sheds light on how the spaceflight environment could affect ureolysis and nitrification processes from a transcriptomic perspective.

Whole transcriptome analysis highlights nutrient limitation of nitrogen cycle bacteria in simulated microgravity.

Regenerative life support systems (RLSS) will play a vital role in achieving self-sufficiency during long-distance space travel. Urine conversion into a liquid nitrate-based fertilizer is a key process in most RLSS. This study describes the effects of simulated microgravity (SMG) on Comamonas testosteroni, Nitrosomonas europaea, Nitrobacter winogradskyi and a tripartite culture of the three, in the context of nitrogen recovery for the Micro-Ecological Life Support System Alternative (MELiSSA). Rotary cell culture systems (RCCS) and random positioning machines (RPM) were used as SMG analogues. The transcriptional responses of the cultures were elucidated. For CO2-producing C. testosteroni and the tripartite culture, a PermaLifeTM PL-70 cell culture bag mounted on an in-house 3D-printed holder was applied to eliminate air bubble formation during SMG cultivation. Gene expression changes indicated that the fluid dynamics in SMG caused nutrient and O2 limitation. Genes involved in urea hydrolysis and nitrification were minimally affected, while denitrification-related gene expression was increased. The findings highlight potential challenges for nitrogen recovery in space.

Steerable isobutyric and butyric acid production from CO2 and H2 by Clostridium luticellarii.

Clostridium luticellarii is a recently discovered acetogen that is uniquely capable of producing butyric and isobutyric acid from various substrates (e.g. methanol), but it is unclear which factors influence its (iso)butyric acid production from H2 and CO2 . We aimed to investigate the autotrophic metabolism of C. luticellarii by identifying the necessary growth conditions and examining the effects of pH and metabolite levels on product titers and selectivity. Results show that autotrophic growth of C. luticellarii requires the addition of complex nutrient sources and the absence of shaking conditions. Further experiments combined with thermodynamic calculations identified pH as a key parameter governing the direction of metabolic fluxes. At circumneutral pH (~6.5), acetic acid is the sole metabolic end product but C. luticellarii possesses the unique ability to co-oxidize organic acids such as valeric acid under high H2 partial pressures (>1 bar). Conversely, mildly acidic pH (≤5.5) stimulates the production of butyric and isobutyric acid while partly halting the oxidation of organic acids. Additionally, elevated acetic acid concentrations stimulated butyric and isobutyric acid production up to a combined selectivity of 53 ± 3%. Finally, our results suggest that isobutyric acid is produced by a reversible isomerization of butyric acid, but valeric and caproic acid are not isomerized. These combined insights can inform future efforts to optimize and scale-up the production of valuable chemicals from CO2 using C. luticellarii.

The nutritional composition and cell size of microbial biomass for food applications are defined by the growth conditions.

BACKGROUND: It is increasingly recognized that conventional food production systems are not able to meet the globally increasing protein needs, resulting in overexploitation and depletion of resources, and environmental degradation. In this context, microbial biomass has emerged as a promising sustainable protein alternative. Nevertheless, often no consideration is given on the fact that the cultivation conditions affect the composition of microbial cells, and hence their quality and nutritional value. Apart from the properties and nutritional quality of the produced microbial food (ingredient), this can also impact its sustainability. To qualitatively assess these aspects, here, we investigated the link between substrate availability, growth rate, cell composition and size of Cupriavidus necator and Komagataella phaffii. RESULTS: Biomass with decreased nucleic acid and increased protein content was produced at low growth rates. Conversely, high rates resulted in larger cells, which could enable more efficient biomass harvesting. The proteome allocation varied across the different growth rates, with more ribosomal proteins at higher rates, which could potentially affect the techno-functional properties of the biomass. Considering the distinct amino acid profiles established for the different cellular components, variations in their abundance impacts the product quality leading to higher cysteine and phenylalanine content at low growth rates. Therefore, we hint that costly external amino acid supplementations that are often required to meet the nutritional needs could be avoided by carefully applying conditions that enable targeted growth rates. CONCLUSION: In summary, we demonstrate tradeoffs between nutritional quality and production rate, and we discuss the microbial biomass properties that vary according to the growth conditions.

Trophic interactions shape the spatial organization of medium-chain carboxylic acid producing granular biofilm communities.

Granular biofilms producing medium-chain carboxylic acids (MCCA) from carbohydrate-rich industrial feedstocks harbor highly streamlined communities converting sugars to MCCA either directly or via lactic acid as intermediate. We investigated the spatial organization and growth activity patterns of MCCA producing granular biofilms grown on an industrial side stream to test (i) whether key functional guilds (lactic acid producing Olsenella and MCCA producing Oscillospiraceae) stratified in the biofilm based on substrate usage, and (ii) whether spatial patterns of growth activity shaped the unique, lenticular morphology of these biofilms. First, three novel isolates (one Olsenella and two Oscillospiraceae species) representing over half of the granular biofilm community were obtained and used to develop FISH probes, revealing that key functional guilds were not stratified. Instead, the outer 150-500 µm of the granular biofilm consisted of a well-mixed community of Olsenella and Oscillospiraceae, while deeper layers were made up of other bacteria with lower activities. Second, nanoSIMS analysis of 15N incorporation in biofilms grown in normal and lactic acid amended conditions suggested Oscillospiraceae switched from sugars to lactic acid as substrate. This suggests competitive-cooperative interactions may govern the spatial organization of these biofilms, and suggests that optimizing biofilm size may be a suitable process engineering strategy. Third, growth activities were similar in the polar and equatorial biofilm peripheries, leaving the mechanism behind the lenticular biofilm morphology unexplained. Physical processes (e.g., shear hydrodynamics, biofilm life cycles) may have contributed to lenticular biofilm development. Together, this study develops an ecological framework of MCCA-producing granular biofilms that informs bioprocess development.

Plant and microbial science and technology as cornerstones to Bioregenerative Life Support Systems in space.

Long-term human space exploration missions require environmental control and closed Life Support Systems (LSS) capable of producing and recycling resources, thus fulfilling all the essential metabolic needs for human survival in harsh space environments, both during travel and on orbital/planetary stations. This will become increasingly necessary as missions reach farther away from Earth, thereby limiting the technical and economic feasibility of resupplying resources from Earth. Further incorporation of biological elements into state-of-the-art (mostly abiotic) LSS, leading to bioregenerative LSS (BLSS), is needed for additional resource recovery, food production, and waste treatment solutions, and to enable more self-sustainable missions to the Moon and Mars. There is a whole suite of functions crucial to sustain human presence in Low Earth Orbit (LEO) and successful settlement on Moon or Mars such as environmental control, air regeneration, waste management, water supply, food production, cabin/habitat pressurization, radiation protection, energy supply, and means for transportation, communication, and recreation. In this paper, we focus on air, water and food production, and waste management, and address some aspects of radiation protection and recreation. We briefly discuss existing knowledge, highlight open gaps, and propose possible future experiments in the short-, medium-, and long-term to achieve the targets of crewed space exploration also leading to possible benefits on Earth.

Predicting the performance of chain elongating microbiomes through flow cytometric fingerprinting.

As part of the circular bio-economy paradigm shift, waste management and valorisation practices have moved away from sanitation and towards the production of added-value compounds. Recently, the development of mixed culture bioprocess for the conversion of waste(water) to platform chemicals, such as medium chain carboxylic acids, has attracted significant interest. Often, the microbiology of these novel bioprocesses is less diverse and more prone to disturbances, which can lead to process failure. This issue can be tackled by implementing an advanced monitoring strategy based on the microbiology of the process. In this study, flow cytometry was used to monitor the microbiology of lactic acid chain elongation for the production of caproic acid, and assess its performance both qualitatively and quantitatively. Two continuous stirred tank reactors for chain elongation were monitored flow cytometrically for over 336 days. Through community typing, four specific community types could be identified and correlated to both a specific functionality and genotypic diversity. Additionally, the machine-learning algorithms trained in this study demonstrated the ability to predict production rates of, amongst others, caproic acid with high accuracy in the present (R² > 0.87) and intermediate accuracy in the near future (R² > 0.63). The identification of specific community types and the development of predictive algorithms form the basis of advanced bioprocess monitoring based on flow cytometry, and have the potential to improve bioprocess control and optimization, leading to better product quality and yields.

RNA extraction protocol from low-biomass bacterial Nitrosomonas europaea and Nitrobacter winogradskyi cultures for whole transcriptome studies.

RNA-sequencing for whole transcriptome analysis requires high-quality RNA in adequate amounts, which can be difficult to generate with low-biomass-producing bacteria where sample volume is limited. We present an RNA extraction protocol for low-biomass-producing autotrophic bacteria Nitrosomonas europaea and Nitrobacter winogradskyi cultures. We describe steps for sample collection, lysozyme-based enzymatic lysis, and a commercial silica-column-based RNA extraction. We then detail evaluation of RNA yield and quality for downstream applications such as RNA-Seq. For complete details on the use and execution of this protocol, please refer to Verbeelen et al.1.

Thermophilic caproic acid production from grass juice by sugar-based chain elongation.

Medium chain carboxylic acids (MCCA) such as caproic acid have a plethora of applications, ranging from food additives to bioplastics. MCCA can be produced via microbial chain elongation using waste and side-streams as substrates, a process that can be more sustainable than conventional production routes. Most chain elongation studies have focused on mesophilic conditions, with only two recent studies hinting at the possibility of thermophilic chain elongation, but a systematic study of its mechanisms is lacking. Here, we investigated thermophilic chain elongation from grass juice, to understand the effect of key operational parameters (pH, temperature, substrate) on the process performance and to establish the key microbial genera and their role in the system. The genus Caproiciproducens was identified as responsible for thermophilic chain elongation, and caproic acid production was most favorable at pH 6.0 and 50 °C among the conditions tested, reaching an average concentration of 3.4 g/L. Batch experiments showed that the substrate for caproic acid production were glucose and xylose, while lactic acid led to the production of only butyric acid. Fed-batch experiments showed that substrate availability and the presence of caproic acid in the system play a major role in shaping the profile of thermophilic chain elongation. The increase of the total sugar concentration by glucose addition (without changing the organic load) during continuous operation led to a microbial community dominated (75 %) by Caproiciproducens and increased by 76 % the final average caproic acid concentration to 6.0 g/L (13 gCOD/L) which represented 32 % (g/g) of the total carboxylic acids. The highest concentration achieved was 7.2 g/L (day 197) which is the highest concentration reported under thermophilic conditions thus far. The results of this work pave the way to the potential development of thermophilic systems for upgrading various underexplored abundant and cheap sugar-rich side-streams to caproic acid.

Why do lactic acid bacteria thrive in chain elongation microbiomes?

Efficient waste management is necessary to transition towards a more sustainable society. An emerging trend is to use mixed culture biotechnology to produce chemicals from organic waste. Insights into the metabolic interactions between community members and their growth characterization are needed to mediate knowledge-driven bioprocess development and optimization. Here, a granular sludge bioprocess for the production of caproic acid through sugar-based chain elongation metabolism was established. Lactic acid and chain-elongating bacteria were identified as the two main functional guilds in the granular community. The growth features of the main community representatives (isolate Limosilactobacillus musocae G03 for lactic acid bacteria and type strain Caproiciproducens lactatifermentans for chain-elongating bacteria) were characterized. The measured growth rates of lactic acid bacteria (0.051 ± 0.005 h-1) were two times higher than those of chain-elongating bacteria (0.026 ± 0.004 h-1), while the biomass yields of lactic acid bacteria (0.120 ± 0.005 g biomass/g glucose) were two times lower than that of chain-elongating bacteria (0.239 ± 0.007 g biomass/g glucose). This points towards differential growth strategies, with lactic acid bacteria resembling that of a r-strategist and chain-elongating bacteria resembling that of a K-strategist. Furthermore, the half-saturation constant of glucose for L. mucosae was determined to be 0.35 ± 0.05 g/L of glucose. A linear trend of caproic acid inhibition on the growth of L. mucosae was observed, and the growth inhibitory caproic acid concentration was predicted to be 13.6 ± 0.5 g/L, which is the highest reported so far. The pre-adjustment of L. mucosae to 4 g/L of caproic acid did not improve the overall resistance to it, but did restore the growth rates at low caproic acid concentrations (1-4 g/L) to the baseline values (i.e., growth rate at 0 g/L of caproic acid). High resistance to caproic acid enables lactic acid bacteria to persist and thrive in the systems intended for caproic acid production. Here, insights into the growth of two main functional guilds of sugar-based chain elongation systems are provided which allows for a better understanding of their interactions and promotes future bioprocess design and optimization.

Optimization of RNA extraction for bacterial whole transcriptome studies of low-biomass samples.

We developed a procedure for extracting maximal amounts of high-quality RNA from low-biomass producing (autotrophic) bacteria for experiments where sample volume is limited. Large amounts of high-quality RNA for downstream analyses cannot be obtained using larger quantities of culture volume. The performance of standard commercial silica-column based kit protocols and these procedures amended by ultrasonication or enzymatic lysis were assessed. The ammonium-oxidizing Nitrosomonas europaea and nitrite-oxidizing Nitrobacter winogradskyi were used as model organisms for optimization of the RNA isolation protocol. Enzymatic lysis through lysozyme digestion generated high-quality, high-yield RNA samples. Subsequent RNA-seq analysis resulted in qualitative data for both strains. The RNA extraction procedure is suitable for experiments with volume and/or biomass limitations, e.g., as encountered during space flight experiments. Furthermore, it will also result in higher RNA yields for whole transcriptome experiments where sample volume and/or biomass was increased to compensate the low-biomass characteristic of autotrophs.

Ammonia oxidation by novel "Candidatus Nitrosacidococcus urinae" is sensitive to process disturbances at low pH and to iron limitation at neutral pH.

Acid-tolerant ammonia-oxidizing bacteria (AOB) can open the door to new applications, such as partial nitritation at low pH. However, they can also be problematic because chemical nitrite oxidation occurs at low pH, leading to the release of harmful nitrogen oxide gases. In this publication, the role of acid-tolerant AOB in urine treatment was explored. On the one hand, the technical feasibility of ammonia oxidation under acidic conditions for source-separated urine with total nitrogen concentrations up to 3.5 g-N L-1 was investigated. On the other hand, the abundance and growth of acid-tolerant AOB at more neutral pH was explored. Under acidic conditions (pH of 5), ammonia oxidation rates of 500 mg-N L-1 d-1 and 10 g-N g-VSS-1 d-1 were observed, despite high concentrations of 15 mg-N L-1 of the AOB-inhibiting compound nitrous acid and low concentration of 0.04 mg-N L-1 of the substrate ammonia. However, ammonia oxidation under acidic conditions was very sensitive to process disturbances. Even short periods of less than 12 h without oxygen or without influent resulted in a complete cessation of ammonia oxidation with a recovery time of up to two months, which is a problem for low maintenance applications such as decentralized treatment. Furthermore, undesirable nitrogen losses of about 10% were observed. Under acidic conditions, a novel AOB strain was enriched with a relative abundance of up to 80%, for which the name "Candidatus (Ca.) Nitrosacidococcus urinae" is proposed. While Nitrosacidococcus members were present only to a small extent (0.004%) in urine nitrification reactors operated at pH values between 5.8 and 7, acid-tolerant AOB were always enriched during long periods without influent, resulting in an uncontrolled drop in pH to as low as 2.5. Long-term experiments at different pH values showed that the activity of "Ca. Nitrosacidococcus urinae" decreased strongly at a pH of 7, where they were also outcompeted by the acid-sensitive AOB Nitrosomonas halophila. The experiment results showed that the decreased activity of "Ca. Nitrosacidococcus urinae" correlated with the limited availability of dissolved iron at neutral pH.

High-rate conversion of lactic acid-rich streams to caproic acid in a fermentative granular system.

Lactic acid-driven chain elongation enables upgrading low-value organic streams into caproic acid. Recently, volumetric production rates over 0.5 g L-1 h-1have been reported for carbohydrate-rich streams in expanded granular sludge bed (EGSB) reactors. However, many target streams contain mixtures of carbohydrates and lactic acid, and little is known about their impact on product profile and microbial ecology, or the importance of carbohydrates as substrate to achieve high rates. This manuscript investigated varying glucose-to-lactate ratios and observed that decreasing glucose-content eliminated odd-chain by-products, while glucose omission required acetic acid addition to support lactic acid conversion. Decreasing the glucose-content fed resulted in decreasing amounts of granular biomass, with the disappearance of granules when no glucose was fed. Lowering the HRT to 0.3 days while feeding only lactic and acetic acid likely triggered re-granulation, enabling the highest lactic acid-driven caproic acid production rates reported thus far at 16.4 ± 1.7 g L-1 d-1.

Pre-incubation conditions determine the fermentation pattern and microbial community structure in fermenters at mild hydrostatic pressure.

Fermentation at elevated hydrostatic pressure is a novel strategy targeting product selectivity. However, the role of inoculum history and cross-resistance, that is, acquired tolerance from incubation under distinctive environmental stress, remains unclear in high-pressure operation. In our here presented work, we studied fermentation and microbial community responses of halotolerant marine sediment inoculum (MSI) and anaerobic digester inoculum (ADI), pre-incubated in serum bottles at different temperatures and subsequently exposed to mild hydrostatic pressure (MHP; < 10 MPa) in stainless steel reactors. Results showed that MHP effects on microbial growth, activity, and community structure were strongly temperature-dependent. At moderate temperature (20°C), biomass yield and fermentation were not limited by MHP; suggesting a cross-resistance effect from incubation temperature and halotolerance. Low temperatures (10°C) and MHP imposed kinetic and bioenergetic limitations, constraining growth and product formation. Fermentation remained favorable in MSI at 28°C and ADI at 37°C, despite reduced biomass yield resulting from maintenance and decay proportionally increasing with temperature. Microbial community structure was modified by temperature during the enrichment, and slight differences observed after MHP-exposure did not compromise functionality. Results showed that the relation incubation temperature-halotolerance proved to be a modifier of microbial responses to MHP and could be potentially exploited in fermentations to modulate product/biomass ratio.

Methylotrophs: from C1 compounds to food.

Microbial protein (MP) is back on the table after decades of slumbering interest. One-carbon (C1) substrates are attractive for MP production due to their efficient production from CO2 and renewable electricity, linking carbon capture to food while circumventing agriculture. Here we compared all reported combinations of C1 (formate/methanol/methane) and microorganisms (bacteria/yeasts) in terms of engineering and biomass quality parameters, focusing on the amino acid match with human requirements. This meta-analysis based on >100 studies suggests that methanol is the most promising C1, and methanol-grown microorganisms seem most nutritional with bacteria and yeasts having different merits. More sustainable MP could be produced if metabolic engineering tools yielding microorganisms with more efficient C1 assimilation pathways and steered amino acid profiles are deployed.

Development of Nitrogen Recycling Strategies for Bioregenerative Life Support Systems in Space.

To enable long-distance space travel, the development of a highly efficient and robust system to recover nutrients from waste streams is imperative. The inability of the current physicochemical-based environmental control and life support system (ECLSS) on the ISS to produce food in situ and to recover water and oxygen at high enough efficiencies results in the need for frequent resupply missions from Earth. Therefore, alternative strategies like biologically-based technologies called bioregenerative life support systems (BLSSs) are in development. These systems aim to combine biological and physicochemical processes, which enable in situ water, oxygen, and food production (through the highly efficient recovery of minerals from waste streams). Hence, minimalizing the need for external consumables. One of the BLSS initiatives is the European Space Agency's (ESA) Micro-Ecological Life Support System Alternative (MELiSSA). It has been designed as a five-compartment bioengineered system able to produce fresh food and oxygen and to recycle water. As such, it could sustain the needs of a human crew for long-term space exploration missions. A prerequisite for the self-sufficient nature of MELiSSA is the highly efficient recovery of valuable minerals from waste streams. The produced nutrients can be used as a fertilizer for food production. In this review, we discuss the need to shift from the ECLSS to a BLSS, provide a summary of past and current BLSS programs and their unique approaches to nitrogen recovery and processing of urine waste streams. In addition, compartment III of the MELiSSA loop, which is responsible for nitrogen recovery, is reviewed in-depth. Finally, past, current, and future related ground and space demonstration and the space-related challenges for this technology are considered.

Electrochemical In Situ pH Control Enables Chemical-Free Full Urine Nitrification with Concomitant Nitrate Extraction.

Urine is a valuable resource for nutrient recovery. Stabilization is, however, recommended to prevent urea hydrolysis and the associated risk for ammonia volatilization, uncontrolled precipitation, and malodor. This can be achieved by alkalinization and subsequent biological conversion of urea and ammonia into nitrate (nitrification) and organics into CO2. Yet, without pH control, the extent of nitrification is limited as a result of insufficient alkalinity. This study explored the feasibility of an integrated electrochemical cell to obtain on-demand hydroxide production through water reduction at the cathode, compensating for the acidification caused by nitritation, thereby enabling full nitrification. To deal with the inherent variability of the urine influent composition and bioprocess, the electrochemical cell was steered via a controller, modulating the current based on the pH in the bioreactor. This provided a reliable and innovative alternative to base addition, enabling full nitrification while avoiding the use of chemicals, the logistics associated with base storage and dosing, and the associated increase in salinity. Moreover, the electrochemical cell could be used as an in situ extraction and concentration technology, yielding an acidic concentrated nitrate-rich stream. The make-up of the end product could be tailored by tweaking the process configuration, offering versatility for applications on Earth and in space.

Chain elongators, friends, and foes.

Bioproduction of medium chain carboxylic acids has recently emerged as an alternative strategy to valorize low-value organic waste and side-streams. Key to this route is chain elongation, an anaerobic microbial process driven by ethanol, lactic acid, or carbohydrates. Because these technologies use wastes as feedstocks, mixed microbial communities are often considered as biocatalysts. Understanding and steering these microbiomes is key to optimize bioprocess performance. From a meta-analysis of publicly available sequencing data, we (i) explore how the current collection of isolated chain elongators compares to microbiome members, (ii) discuss the main beneficial and antagonistic interactions with community partners, and (iii) identify the key research gaps and needs to help understand chain elongation microbiomes, and design/steer these novel bioproduction processes.